1. Field of the Invention
This invention relates to enzyme immunoassays whose sensitivities are greatly increased by cascade amplification.
2. Description of the Prior Art
Single-stage enzyme amplified immunoassays are well known and are disclosed in books such as "immunoassays in the Clinical Laboratory", Nakamura, et al., (ed.) Alan R. Liss, Inc., N.Y.C. (pub.) (1979); "immunoassays-Clinical Laboratory Techniques for the 1980's", Nakamura, et al., (ed.), Alan R. Liss, Inc., N.Y.C. (pub.), (1980); and "The Tools of Biochemistry", Cooper, John Wiley & Sons, N.Y.C. (pub.), (1977); among others.
U.S. Pat. No. 3,654,090 (Schuurs, et al.,) discloses enzyme assays for the determination of antigens and antibodies, comprising using a reagent consisting of one component of an antigen-antibody reaction in an insolubilzed form and the other one cavalently linked to an enzyme. There is further disclosure of quantitative assays using colorimetry, spectrophotometry, fluorospectrophotometry or gaseometry, and qualitative assays using either a colored substrate or a colored end product. At column 2, lines 32-42 it is stated that: "Further possibilities are: The conjugated enzyme generates the substrate for a second enzyme, which gives a coloured end-product. The conjugated enzyme converts a pro-enzyme into an enzyme, which catalyses a reaction with a coloured compound involved. The conjugated enzyme catalyses a reaction wherein substrate or end-product can be stained easily. Many enzymes can be used in reactions as described above, such as peroxidase, .beta.-glucuronidase, .beta.-D-glucosidase, .beta.-D-galactosidase, urease, glucose oxides + peroxidase, and galactose oxidase + peroxidase".
U.S. Pat. No. 3,966,556 (Rubenstein, et al.,) [and related U.S. Pat. Nos. 3,817,837; 3,875,011; 4,190,496; 4,191,613; and 4,203,802] disclose enzyme assays in which an enzyme is bound to a ligand or ligand counterfeit. A receptor when bound to the enzyme-bound-ligand substantially inhibits enzymatic activity, providing for different catalytic efficiences of enzyme-bound-ligand with and without the receptor. There is no disclosure of cascade amplification.
Other U.S. patents that disclose enzyme assays (but without cascade amplification) include: U.S. Pat. Nos. 3,791,932; 3,839,153; 3,850,752; 4,002,532; 4,043,872; 4,134,792; 4,228,237; and 4,238,565.
Also well known, is the general concept of the "cascade phenomenon", whereby there is a sequential activation of enzymatic components. The best known example of an enzymatic cascade is the process of blood clotting [see, for example, "Basic Physiology for the Health Sciences", Selkart (ed.), Little, Brown and Company, Boston (pub.) (1975) at pages 340-345; and "Human Blood coagulation, Haemostasis and Thrombosis", Biggs (ed.), 2nd ed., Blackwell Scientific Publications, London (pub.) (1976)]. In "Human Blood Coagulation, etc.", supra, there is disclosed at pages 49-54 an assay for measuring prothrombin comprising activation of the proenzyme prothrombin to the enzyme thrombin by thrombokinase, and the subsequent conversion of fibrinogen to fibrin by the thrombin. Since enzymes are true catalysts and each enzyme molecule acts on a number of molecules causing a multiplier effect, a two stage cascade causes a geometric increase in the molecules that are acted upon. In "Enzymatic Reaction Mechanisms", Walsh, W. H. Freeman and Company, San Francisco (pub.) (1979) at pages 116-170, there is the statement: "This process is called a cascade phenomenon (sequential activation of components), and this is only one example of many such phenomena in enzymatic systems (the process of blood clotting and the mobilization of glycogen being two others) where such complex sequences operate. It has been argued that cascade systems are sensitive ways of amplifying an initial small biological signal. Because each component in the cascade is a catalytic entity, one can have large multiplication factors at each step".
"Methods in Enzymology" XLV, Lorand (ed.), Academic Press (pub.) (1976) at pages 239-273 discloses the activation of plasminogen to plasmin by urokinase (pp. 242-243) and by streptokinase (pp. 245-246); and the use of caseinolytic and fibrinolytic substrates (pp. 257-258). In Thrombosis Research, 13:733-729 (1978) there is disclosed a fluorescent substrate assay for plasminogen after its conversion to plasmin by streptokinase. The specific fluorescent substrate disclosed is H-D-valine-leucine-lysine-5-amidoisophtholic acid dimethyl ester di-tri-fluoroacetate, and comparison was made with a caseinolytic assay and a radial immunodiffusion assay. In Proc. Natl. Acad. Sci. USA 75:750-753 (1978) a similar assay is disclosed using a synthetic fluorogenic peptide substrate, 7-(N-Cbz-Glycylglycylarginin-amido)-i-methylcoumarin trifluoroacetate. In Thrombosis Research 13:389-395 (1978) there is disclosed a solid phase assay for fibrinogen activators (i.e., streptokinase and urokinase) using .sup.125 I labeled fibrinogen. There is no disclosure of the coupling of ligands to the fibrinogen. In Comp. and Biomed. Res. 11:119-132 (1978) there is a discussion of blood clotting enzyme cascades in connection with a plasminogen-plasmin assay using streptokinase as the activator.